The present invention relates to novel methods and devices for detecting whole or non-fragmented parathyroid hormone (wPTH) in a biological sample. In particular, a novel monoclonal or polyclonal antibody or antibody fragment is used that is specific for a portion of the initial peptide sequence for wPTH which comprises a domain for adenylate cyclase activation, (amino acids 2 to 8), wherein at least four amino acids are part of the reactive portion with the antibody.
Calcium plays an indispensable role in cell permeability, the formation of bones and teeth, blood coagulation, transmission of nerve impulse, and normal muscle contraction. The concentration of calcium ions in the blood is, along with calcitrot and calcitonin, regulated mainly by parathyroid hormone (PTH). Although calcium intake and excretion may vary, PTH serves through a feedback mechanism to maintain a steady concentration of calcium in cells and surrounding fluids. When serum calcium lowers, the parathyroid glands secrete PTH, affecting the release of stored calcium. When serum calcium increases, stored calcium release is retarded through lowered secretions of PTH.
The complete form of human PTH, (hPTH), is a unique 84 amino acid peptide (SEQ ID NO. 2), as is shown in FIG. 1. Researchers have found that this peptide has an anabolic effect on bone that involves a domain for protein kinase C activation (amino acid residues 28 to 34) as well as a domain for adenylate cyclase activation (amino acid residues 1 to 7). However, various catabolic forms of clipped or fragmented PTH peptides also are found in circulation, most likely formed by intraglandular or peripheral metabolism. For example, whole PTH can be cleaved between amino acids 34 and 35 to produce a (1-34) PTH N-terminal fragment and a (35-84) PTH C-terminal fragment. Likewise, clipping can occur between either amino acids 36 and 37 or 37 and 38. Recently, a large PTH fragment referred to as xe2x80x9cnon-(1-84) PTHxe2x80x9d has been disclosed which is clipped closer to the N-terminal end of PTH. (See R. LePage el alia, xe2x80x9cA non-(1-84) circulating parathyroid hormone (PTH) fragment interferes significantly with intact PTH commercial assay measurements in uremic samples xe2x80x9d Clin Chem (1998); 44: 805-810.)
The clinical need for accurate measurement of PTH is well demonstrated. Serum PTH level is one of the most important index for patients with the following diseases: familial hypocalciuric hypercalcemia; multiple endocrine neoplasia types I and II; osteoporosis; Paget""s bone disease; primary hyperparathyroidismxe2x80x94caused by primary hyperplasia or adenoma of the parathyroid glands; pseudohypoparathyroidism, and renal failure, which can cause secondary hyperparathyroidism.
Determining circulating biologically active PTH levels in humans has been challenging. One major problem is that PTH is found at low levels, normally 10 pg/mL to 40 pg/mL. Coupled with extremely low circulating levels is the problem of the heterogeneity of PTH and its many circulating fragments. In many cases, immunoassays have faced substantial and significant interference from circulating PTH fragments. For example, some commercially available PTH kits have almost 100% cross-reactivity with the non-( 1-84) PTH fragment, (see the LePage article).
PTH immunoassays have varied over the years. One early approach is a double antibody precipitation immunoassay found in U.S. Pat. No. 4,369,138 to Arnold W. Lindall el alia. A first antibody has a high affinity for a (65-84) PTH fragment. A radioactive labeled (65-84) PTH peptide is added to the sample with the first antibody to compete for the unlabeled peptide. A second antibody is added which binds to any first antibody and radioactive labeled PTH fragment complex, thereby forming a precipitate. Both precipitate and supernatant can be measured for radioactive activity, and PTH levels can be calculated therefrom.
In an effort to overcome PTH fragment interference, immunoradiometric two-site assays for intact PTH (I-PTH) have been introduced, such as Allegro(copyright) Intact PTH assay by the Nichols Institute of San Juan Capistrano, Calif. In one version, a capture antibody specifically binds to the C-terminal portion of hPTH while a labeled antibody specifically binds to the N-terminal portion of the captured hPTH. In another, two monoclonal antibodies were used, both of which attached to the N-terminal portion of hPTH. (For the purposes of the present invention, the complete form of human PTH is referred to as xe2x80x9cwhole PTRxe2x80x9d or xe2x80x9cwPTHxe2x80x9d as distinguished from xe2x80x9cintact PTHxe2x80x9d or xe2x80x9cI-PTHxe2x80x9d which can include not only wPTH, but also a large PTH fragment cleaved about amino acids 5 to 8.) Unfortunately, these assays have problems in that they measure but do not discriminate between w-PTH and I-PTH. This inability comes to the fore in hyperparathyroid patients and renal failure patients who have significant endogenous concentrations of large, non-whole PTH fragments.
Recently, researchers have made a specific binding assay directed to the large N-terminal PTH fragments. (See. Gao, Ping et alia xe2x80x9cImmunochemicalluminometric assay with two monoclonal antibodies against the N-terminal sequence of human parathyroid hormonexe2x80x9d, Clinica Chimica Acta 245 (1996) 39-59.) This immunochemiluminometric assay uses two monoclonal antibodies to detect N-terminal (1-34) PTH fragments but not mid-portion PTH fragments or C-terminal PTH fragments. A key factor in the design of these assays is to eliminate any reaction with C-terminal PTH fragments.
The present invention relates to a method for detecting wPTH in a biological sample without detecting the non (1-84) large PTH fragment component of I-PTH, and in particular to a substantially pure monoclonal or polyclonal antibody or antibody fragment specific for the initial sequence for wPTH which comprises a domain for adenylate cyclase activation, VAL-SER-GLU-ILE-GLN-LEU-MET (SEQ ID NO. 3), wherein at least four amino acids in this sequence are part of the antibody reactive portion of the peptide. The method for measuring the amount of wPTH in a sample such as serum, plasma, or blood comprises four general steps which can vary depending upon whether one uses a first antibody or antibody fragment specific for the PTH peptide VAL-SER-GLU-ILE-GLN-LEU-MET (SEQ ID NO. 3), wherein at least four amino acids are part of the antibody reactive portion of the peptide either as a signal antibody or a capture antibody in conventional immunoassay formats. Used either as a signal antibody or as a capture antibody, enough antibody is added to bind all w-PTH present. Next, one allows the first antibody to bind to any wPTH present, thereby forming a complex. A specific binding label comprised of a second antibody and a conventional immunoassay label, such as chemiluminescent agents, colorimetric agents, energy transfer agents, enzymes, fluorescent agents, and radioisotopes, is used to label the complex, preferably at the N-terminal end of wPTH, and can be added either substantially simultaneously with the first antibody or subsequent thereto. Finally, one uses conventional techniques to measure the amount of labeled complex, and thereby calculate wPTH levels in the sample. If used as a signal antibody, then the first.
One can also use the present invention to detect the amount of N-terminal PTH fragment present having a complete and functional amino acid sequence from 1 to at least 34. Some researchers are using synthetic N-terminal PTH peptides as a therapeutic treatment to stop bone loss and actually encourage an increase in bone mass. These peptides range from amino acids 1 to at least 34 and on up to 38. The present assay can detect the amount and duration of such synthetic peptides in circulation in a patient.